A second gene, VpreB in the lambda 5 locus of the mouse, which appears to be selectively expressed in pre-B lymphocytes.

The murine gene lambda 5 is selectively expressed in pre-B lymphocytes. Of the three exons encoding lambda 5, exons II and III show strong homologies to immunoglobulin lambda light (L) chain gene segments, i.e. to J lambda intron and exon, and C lambda exon sequences respectively. We have now found, 4.6 kb upstream of lambda 5, another gene composed of two exons which is selectively expressed in pre-B cell lines as a 0.85 kb mRNA potentially coding for a protein of 142 amino acids including a 19 amino acid-long signal peptide. The 5' sequences of this gene show homologies to sequences encoding the variable regions of kappa and lambda L chains and of heavy (H) chains. The deduced amino acid sequence contains the consensus cysteine residues as well as other consensus amino acids at positions which characterize immunoglobulin (Ig) domains. We call the second gene VpreB. The 3' end of VpreB encoding the 26 carboxyl terminal amino acids shows no homology to any known nucleotide sequence. The putative protein encoded by VpreB is a potential candidate for association with the putative protein encoded by lambda 5, and thereby a candidate for association with H chains in pre-B cells. Southern blot analysis of DNA from liver (germ line) and 70Z/3 pre-B cell lines reveals two genes which hybridize to the VpreB gene. We call VpreB1 the gene which is found 5' of lambda 5. The other gene, called VpreB2, which has not yet been located within the genome, shows 97% nucleotide sequence homology to VpreB1 in an area of 1 kb which covers the coding region of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two induced anti-Z-DNA monoclonal antibodies use VH gene segments related to those of anti-DNA autoantibodies

The cDNA for H and L chain V regions of two anti-Z-DNA mAb, Z22 and Z44, were cloned and sequenced. These are the first experimentally induced anti-nucleic acid antibody sequences available for comparison with autoantibody sequences. Z22 and Z44 are IgG2b and IgG2a antibodies from C57BL/6 mice. They recognize different facets of the Z-DNA structure. They both use VH10 family genes and share 95% sequence base sequence identity in the VH and leader sequences; however, they differ in the 5'-untranslated region of the VH mRNA, indicating they arise from different germline genes. Both use JH4 segments. They differ from each other very extensively in the CDR3 of both H and L chains. The most closely related H chains in the current GenBank/EMBL data base are two mouse IgG anti-DNA autoantibodies, one from an MRL-lpr/lpr mouse (MRL-DNA4) and one from an NZB/NZW mouse (BV04-01). Z22 and Z44 share 95% sequence identity with these antibodies in the VH segment. In addition, Z22 is identical to MRL-DNA4 at 91% of the positions in the 5'-untranslated region of the H chain mRNA. The two antibodies share 95% base sequence identity in the V kappa segment. The most closely related L chains, with 97 to 98% sequence identity, are the V kappa 10b germline gene for Z22 and the V kappa 10a germ line gene, which is associated with A/J anti-arsonate antibodies and BALB/c anti-ABO blood group substance antibodies, for Z44. Z22 and Z44 share several structural features (similarities in VH, JH, and V kappa) but differ very markedly in the L chain CDR1 and both H and L chain CDR3 sequences; these regions may determine the differences in their specific interactions with Z-DNA.     

The productive gene for alpha-H chain disease protein MAL is highly modified by insertion-deletion processes

alpha-H chain diseases (HCD) is a human lymphoproliferative disorder, characterized by the production of truncated alpha-Ig H chains, without associated L chains. In this study, we have analysed the serum protein, the alpha-HCD mRNA and the rearranged alpha-HCD gene from the leukemic cells of a patient (MAL) with alpha-HCD. The abnormal MAL serum Ig consisted of short alpha 1-chains, lacking VH and CH1 domains (only CH2 and CH3 domains were present). The alpha-HCD mRNA (1.2 kb) was shorter than a normal alpha-mRNA (2 kb); the corresponding cDNA had sequences for the leader, a 84-bp sequence of unknown origin and the CH2 and CH3 exons. The establishment of the sequence of the productive alpha-HCD MAL allele revealed two major deletions; that of the VH region as well as that of the CH1 region. The JH region is altered by multiple mutations, small insertions and a duplication of the psi JH3 region. A large insert (INS1), of 360 bp (containing the 84 bp exon found in the cDNA), replaces the deleted VH region. INS1 is non-Ig related and apparently of nongenomic origin. A large second insert (509 bp), is located between the enhancer and the switch region. Insert2 contains repetitive non-Ig-related sequences and a small Ig-related sequence. All these alterations resulted in an abnormal mRNA, which comprises the leader, a 84-bp alien exon derived from INS1 and the CH2 and CH3 exons of the alpha 1-gene.     

Three monoclonal antibodies to the VHS virus glycoprotein: comparison of reactivity in relation to differences in immunoglobulin variable domain gene sequences

Three monoclonal antibodies (MAbs) to the VHSV G protein were compared in different immunoassays and the variable domain cDNA sequences from the respective immunoglobulin (Ig) genes were determined. One MAb (IP1H3) was non-neutralising and recognised different virus isolates equally well in ELISA. The other two were neutralising and recognised the same or closely related epitopes. One of these two MAbs (3F1H10) was more restricted in its ability to neutralise heterologous VHSV isolates than the other (3F1A2). A semi-quantitative relationship between binding of the two neutralising MAbs in ELISA and their neutralising activity was evident. Binding kinetic analyses by plasmon resonance identified differences in the dissociation rate constant (kd) as a possible explanation for the different reactivity levels of the MAbs. The Ig variable heavy (VH) and light (V kappa) domain gene sequences of the three hybridomas were compared. The inferred amino acid sequence of the two neutralising antibody VH domains differed by three amino acid residues (97% identity) and only one residue difference was evident in the V kappa domains. In contrast, IP1H3 shared only 38 and 39% identity with the 3F1A2 and 3F1H10 VH domains respectively and 49 and 50% identity with the 3F1A2 and 3F1H10 V kappa domains respectively. The neutralising antibodies were produced by hybridomas originating from the same fusion and the high nucleotide sequence homology of the variable Ig gene regions indicated that the plasma cell partners of the hybridomas originated from the same virgin B lymphocyte. The few differences observed in the VH and V kappa amino acid sequences were probably due to somatic mutations arising during affinity maturation and might explain the observed reactivity differences between the two MAbs.     

Chicken immunoglobulin gamma-heavy chains: limited VH gene repertoire, combinatorial diversification by D gene segments and evolution of the heavy chain locus

cDNA clones encoding the variable and constant regions of chicken immunoglobulin (Ig) gamma-chains were obtained from spleen cDNA libraries. Southern blots of kidney DNA show that the variable region sequences of eight cDNA clones reveal the same set of bands corresponding to approximately 30 cross-hybridizing VH genes of one subgroup. Since the VH clones were randomly selected, it is likely that the bulk of chicken H-chains are encoded by a single VH subgroup. Nucleotide sequence determinations of two cDNA clones reveal VH, D, JH and the constant region. The VH segments are closely related to each other (83% homology) as expected for VH or the same subgroup. The JHs are 15 residues long and differ by one amino acid. The Ds differ markedly in sequence (20% homology) and size (10 and 20 residues). These findings strongly indicate multiple (at least two) D genes which by a combinatorial joining mechanism diversify the H-chains, a mechanism which is not operative in the chicken L-chain locus. The most notable among the chicken Igs is the so-called 7S IgG because its H-chain differs in many important aspects from any mammalian IgG. The sequence of the C gamma cDNA reported here resolves this issue. The chicken C gamma is 426 residues long with four CH domains (unlike mammalian C gamma which has three CH domains) and it shows 25% homology to the chicken C mu. The chicken C gamma is most related to the mammalian C epsilon in length, the presence of four CH domains and the distribution of cysteines in the CH1 and CH2 domains. We propose that the unique chicken C gamma is the ancestor of the mammalian C epsilon and C gamma subclasses, and discuss the evolution of the H-chain locus from that of chicken with presumably three genes (mu, gamma, alpha) to the mammalian loci with 8-10 H-chain genes.     

Nucleotide sequence of channel catfish heavy chain cDNA and genomic blot analyses. Implications for the phylogeny of Ig heavy chains

Our prior analyses defined the cDNA sequence on part of the CH2 domain, the complete CH3 and CH4 domains, and the 3'-untranslated region of a catfish H chain. To complete the catfish H chain mRNA sequence, a primer-extended H chain cDNA library was constructed. Analysis of this library has resulted in the definition of full-length clones encoding a 61-bp 5' untranslated region, a 51-bp leader sequence, the V region and the complete CH1 and CH2 domains. The high similarity defined with other vertebrate V regions readily allowed the catfish sequence to be divided into FR and CDR regions. Sequence comparisons with mammalian VH and JH genes strongly suggest that the catfish V region is the product of multiple genes. Using a catfish VH cDNA probe, at least 25 different genomic VH members were defined. Because this probe does not hybridize with other full-length H chain cDNA clones, additional VH families will likely be defined in catfish. Phylogenetic sequence comparisons of the catfish C region domains indicated that the CH1 and CH4 were the most highly conserved. In addition several important features were defined in genomic Southern blot analyses of catfish DNA. Gene titration experiments established that the catfish CH gene is represented by a single genomic copy. This finding provides clear evidence that the genomic organization of H chain genes in catfish must be different from that defined in sharks and suggests that the phylogeny of single copy CH genes may have been established at the level of the bony fishes. It is also likely that there is an additional CH gene in catfish. This gene is also represented by a single genomic copy, and based upon its relative signal intensity when compared with the known CH gene it appears to share higher similarity with the known CH1 domain than it does with the CH2 domain.     

Molecular cloning of the Clostridium botulinum structural gene encoding the type B neurotoxin and determination of its entire nucleotide sequence.

DNA fragments derived from the Clostridium botulinum type A neurotoxin (BoNT/A) gene (botA) were used in DNA-DNA hybridization reactions to derive a restriction map of the region of the C. botulinum type B strain Danish chromosome encoding botB. As the one probe encoded part of the BoNT/A heavy (H) chain and the other encoded part of the light (L) chain, the position and orientation of botB relative to this map were established. The temperature at which hybridization occurred indicated that a higher degree of DNA homology occurred between the two genes in the H-chain-encoding region. By using the derived restriction map data, a 2.1-kb BglII-XbaI fragment encoding the entire BoNT/B L chain and 108 amino acids of the H chain was cloned and characterized by nucleotide sequencing. A contiguous 1.8-kb XbaI fragment encoding a further 623 amino acids of the H chain was also cloned. The 3' end of the gene was obtained by cloning a 1.6-kb fragment amplified from genomic DNA by inverse polymerase chain reaction. Translation of the nucleotide sequence derived from all three clones demonstrated that BoNT/B was composed of 1,291 amino acids. Comparative alignment of its sequence with all currently characterized BoNTs (A, C, D, and E) and tetanus toxin (TeTx) showed that a wide variation in percent homology occurred dependent on which component of the dichain was compared. Thus, the L chain of BoNT/B exhibits the greatest degree of homology (50% identity) with the TeTx L chain, whereas its H chain is most homologous (48% identity) with the BoNT/A H chain. Overall, the six neurotoxins were shown to be composed of highly conserved amino acid domains interceded with amino acid tracts exhibiting little overall similarity. In total, 68 amino acids of an average of 442 are absolutely conserved between L chains and 110 of 845 amino acids are conserved between H chains. Conservation of Trp residues (one in the L chain and nine in the H chain) was particularly striking. The most divergent region corresponds to the extreme carboxy terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.     

Molecular cloning of the Clostridium botulinum structural gene encoding the type B neurotoxin and determination of its entire nucleotide sequence.

DNA fragments derived from the Clostridium botulinum type A neurotoxin (BoNT/A) gene (botA) were used in DNA-DNA hybridization reactions to derive a restriction map of the region of the C. botulinum type B strain Danish chromosome encoding botB. As the one probe encoded part of the BoNT/A heavy (H) chain and the other encoded part of the light (L) chain, the position and orientation of botB relative to this map were established. The temperature at which hybridization occurred indicated that a higher degree of DNA homology occurred between the two genes in the H-chain-encoding region. By using the derived restriction map data, a 2.1-kb BglII-XbaI fragment encoding the entire BoNT/B L chain and 108 amino acids of the H chain was cloned and characterized by nucleotide sequencing. A contiguous 1.8-kb XbaI fragment encoding a further 623 amino acids of the H chain was also cloned. The 3' end of the gene was obtained by cloning a 1.6-kb fragment amplified from genomic DNA by inverse polymerase chain reaction. Translation of the nucleotide sequence derived from all three clones demonstrated that BoNT/B was composed of 1,291 amino acids. Comparative alignment of its sequence with all currently characterized BoNTs (A, C, D, and E) and tetanus toxin (TeTx) showed that a wide variation in percent homology occurred dependent on which component of the dichain was compared. Thus, the L chain of BoNT/B exhibits the greatest degree of homology (50% identity) with the TeTx L chain, whereas its H chain is most homologous (48% identity) with the BoNT/A H chain. Overall, the six neurotoxins were shown to be composed of highly conserved amino acid domains interceded with amino acid tracts exhibiting little overall similarity. In total, 68 amino acids of an average of 442 are absolutely conserved between L chains and 110 of 845 amino acids are conserved between H chains. Conservation of Trp residues (one in the L chain and nine in the H chain) was particularly striking. The most divergent region corresponds to the extreme carboxy terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.     

DNA rearrangements affecting both variable and constant regions of Ig H chain genes in MPC11 mouse myeloma variants

We have examined the mechanisms that account for short Ig H chain production in two variants of the mouse myeloma cell line MPC11 (IgG2b, kappa) by mRNA sequencing and restriction enzyme mapping. One variant, F5.5, has a thymidine residue inserted into the (CH3) domain, of the Ig H chain, resulting in premature termination and translation of a gamma 2b H chain of 50,000 m.w. A second variant, E5.7A12, contains gamma 2a-derived sequences that extend from near the 3' end of the CH2 domain to the intervening sequence between the CH2 and CH3 domains, consistent with a microrecombination event (defined as either a double cross-over or gene conversion event). In this variant, the 5' end of the CH3 domain has been deleted, but the remainder of the gamma 2b(CH3) domain is present, resulting in the translation of a gamma 2b-gamma 2a-gamma 2b H chain of 52,000 m.w. Additional rearrangements affecting sequences in or adjacent to the variable region accompany H chain constant region alterations in these cell lines and subclones of these cell lines. In F5.5, novel sequences have recombined within one of two duplicated copies of the VH gene. In a sister clone of E5.7A12 that has ceased H chain production (E5.7A14), new sequences have recombined within 300 bp 5' of the enhancer element. Both F5.5 and E5.7A12, like their immediate unstable precursor cells, fail to assemble H-H dimers, halting the Ig assembly process at the heavy-light stage, and do not secrete H chains. We speculate that defects in H chain assembly and secretion, as exemplified by the parents of these variants (i.e., intermediates of these secondary variants), reactivate the Ig gene rearrangement machinery and result in the formation of these putatively equally unstable secondary variants.     

Multiple genomic defects result in an alternative RNA splice creating a human gamma H chain disease protein

Heavy chain diseases (HCD) are human lymphoproliferative disorders in which a clonal B cell population produces Ig molecules made of truncated H chains without associated L chain. We characterized the rearranged H chain gene and its mRNA from the leukemic cells of a patient (RIV) with gamma-HCD. The abnormal RIV serum Ig consisted of shortened, dimeric gamma 1-chains which had an amino terminus within the hinge region. RIV lymphoblasts possessed a foreshortened (1200 bp) gamma 1-mRNA which had sequences for only the leader, hinge, second, and third constant region domains (CH2 + CH3), but lacked variable (VH) and CH1 information. Sequence of the productive gamma 1 allele revealed it had undergone VH-JH and H chain class switch recombinations. However, normal RNA splice sites had been eliminated by a DNA insertion/deletion (VH acceptor site), mutations (JH donor site), or a large deletion (CH1 region). Inserted sequences were of non-Ig and apparently non-genomic origin. These DNA alterations resulted in aberrant mRNA processing in which the leader region was spliced directly to the hinge region, accounting for the HCD protein.     

Ig VH, DH, and JH germ-line gene segments linked by overlapping cosmid clones of rabbit DNA

Overlapping cosmid clones of rabbit germ-line DNA containing VH, DH and JH gene segments were isolated. The map of this cluster of cosmid clones indicated that the rabbit VH and JH regions were separated by 63 kb. Hybridization of Southern blots of these cosmid clones with two different DH segment probes identified a total of six DH segments within the region between the VH and JH regions. The nucleotide sequences of the JH region and one of the DH segments have been determined. The DH segment has conserved heptamer and nonamer sequences separated by 12 and 11 bp at the 3' and 5' sides, respectively, of the coding region and hence, appears to be a functional gene. The nucleotide sequence of the JH region revealed four functional JH gene segments and one JH pseudogene. Inasmuch as the JH region had previously been linked by contiguous overlapping clones with C mu, C gamma, C epsilon, and one C alpha gene, this VH-DH-JH cluster and the clones containing the Ig H chain C region genes represent 190 kb of contiguous germ-line DNA of the Ig H chain locus.     

Amino acid sequence of the monomer subunit of the giant multisubunit hemoglobin from the earthworm Pheretima sieboldi

The giant extracellular hemoglobin of the earthworm Pheretima sieboldi is mainly composed of two heme-containing subunits: a monomer; chain I and a disulfide-bonded trimer of chains II, III and IV. Both subunits can be separated easily by gel filtration under alkaline conditions. The amino acid sequence of chain I has been determined. It is composed of 141 residues, has two half-cystine residues forming a intrachain disulfide bridge, and has a molecular mass of 16911 Da including a heme group. Heterogeneity was found at position 37 (His or Ser). The amino acid sequence of Pheretima chain I showed 30-50% identity with those of eight heme-containing chains of Lumbricus and Tylorrhynchus hemoglobins. The sequences of nine chains of annelid giant hemoglobins were compared separately in the functionally essential central exonic region and structurally essential side exonic regions, and a phylogenetic tree was constructed. The amino acid substitution rate for the central exon was found to be about 1.5 times slower than that for the side exons.     

Chicken IgA H chains. Implications concerning the evolution of H chain genes.

A cDNA clone (A1) encoding for a novel chicken Ig H chain isotype was isolated. In sequence comparison to mammalian H chains, A1 C region was most closely related to the alpha isotype. For example, identities of 35%, 32%, and 31% at the amino acid level to rabbit C alpha, C mu, and C gamma were observed, respectively. Distribution of the glucosamine acceptor sites (Asn-X-Ser/Thr) in A1 C region was typical of alpha H chains. Moreover, A1 C region probe hybridized to a 2.2-kb RNA species expressed in the epithelial lymphoid tissues. Thus, A1 was identified as the avian homologue for mammalian alpha H chains. Interestingly, the chicken C alpha was structurally consistent with four complete CH domains, whereas only three domains are present in the mammalian C alpha genes. In addition, interdomain sequence alignments suggested that the homologue for the chicken C alpha 2 domain is missing from the mammalian alpha H chains. Thus, the present data suggest evolution of the IgA isotype before the segregation of avian and mammalian species. Also, the first C alpha gene may have consisted of four CH domains, whereas reorganization of the C alpha 2 region led to the generation of hinge region in the mammalian alpha H chains.     

The nucleotide sequence of the mouse immunoglobulin epsilon gene: comparison with the human epsilon gene sequence

We have determined the nucleotide sequence of the immunoglobulin epsilon gene cloned from newborn mouse DNA. The epsilon gene sequence allows prediction of the amino acid sequence of the constant region of the epsilon chain and comparison of it with sequences of the human epsilon and other mouse immunoglobulin genes. The epsilon gene was shown to be under the weakest selection pressure at the protein level among the immunoglobulin genes although the divergence at the synonymous position is similar. Our results suggest that the epsilon gene may be dispensable, which is in accord with the fact that IgE has only obscure roles in the immune defense system but has an undesirable role as a mediator of hypersensitivity. The sequence data suggest that the human and murine epsilon genes were derived from different ancestors duplicated a long time ago. The amino acid sequence of the epsilon chain is more homologous to those of the gamma chains than the other mouse heavy chains. Two membrane exons, separated by an 80-base intron, were identified 1.7 kb 3' to the CH4 domain of the epsilon gene and shown to conserve a hydrophobic portion similar to those of other heavy chain genes. RNA blot hybridization showed that the epsilon membrane exons are transcribed into two species of mRNA in an IgE hybridoma.     

Sequences of the VH and VL regions of murine monoclonal antibodies against 3-fucosyllactosamine

Many mAb that bind the carbohydrate antigenic determinant 3-fucosyl-lactosamine (3-FL), Gal beta 1-4[Fuc alpha-3]GlcNAc-R have been raised in BALB/c mice, and we are studying the structure and regulation of these antibodies. In this report, we present the first information about their amino acid sequences and the Ig gene segments used to encode them. V regions of the H and L chains of three anti-3-FL antibodies, PMN6, PMN29, and PM81, were sequenced by a combination of mRNA and amino acid sequencing. The L chain sequences of PMN6 and PM81 antibodies indicate that their VK and JK regions are encoded by VK24B and JK1 germ-line genes, respectively. The nucleotide and amino acid sequences of the H chains suggest that the three anti-3-FL antibodies are encoded by the VH441 gene segment of the X24 VH family, and this conclusion was supported by Southern filter hybridization with VH441 and JH3-JH4 probes. PMN29 has at least 11 amino acid substitutions, which is an unusually large amount of somatic mutation for an IgM antibody. Previous analyses of BALB/c genomic libraries with VHX24 and VH441 probes make it unlikely that this VH family contains additional germ-line genes, but this possibility cannot be excluded. All three antibodies use the DQ52 and JH4 gene segments. The single VH and VL gene segments used to encode the anti-3-FL antibodies is in contrast to the multiple VH and VL segments used by antibodies against other carbohydrate Ag such as alpha 1-6 dextran and group A streptococcal carbohydrate. VH441 also encodes the VH regions of antibodies against galactan and levan (beta 2-6 fructosan). The similarities among VH segments of antibodies against 3-FL, levan, and galactan, and the striking differences in their CDR3 sequences, suggest that CDR3 plays an important role in the formation of the Ag binding site. The use of a single VH segment from the smallest VH gene family by antibodies against at least three different carbohydrate determinants is noteworthy. It raises the possibility that the amino acid sequence encoded by VH441 has some general structural features that make it particularly well adapted for binding to carbohydrate sequences.     

Nucleotide sequence of a rabbit IgG heavy chain from the recombinant F-I haplotype

We report the sequence of a cDNA encoding a rabbit immunoglobulin gamma heavy chain of d12 and e14 allotypes with high homology to partial cDNA sequences from rabbits of d11 and e15 allotypes. The encoded rabbit protein shows homologies with human (68-70%) and mouse (60-63%) gamma chains. The nucleotide sequence homologies of the CH domains range from 76-84% with human and 64-76% with mouse sequences. Comparison of the portion of VH encoding amino acid positions 34-112 with a previously determined VH sequence of the same allotype shows high conservation of sequences in the second and third framework segments but more marked differences both in length and encoded amino acids of the second and third complementarity-determining regions (CDRs). We also found a high degree of homology with a human genomic V-region, VH26 (77%) and a remarkable similarity between rabbit and human second CDR sequences and human genomic D minigenes. These results provide additional evidence that D minigene sequences share information with the CDR2 portion of VH regions.     

Immunoglobulin diversity: analysis of the germ-line VH gene repertoire of the murine anti-GAT response.

A cDNA clone was constructed from a mRNA encoding an anti-GAT (Glu60 Ala30 Tyr10) BALB/c monoclonal antibody heavy chain. Its sequence, covering codons -5 to 162 and therefore encompassing the complete V-D-J region, was determined. Surprisingly, the sequence of the VH gene-encoded region was almost identical with that of the BALB/c VH anti-HNP (4-hydroxy-3-nitrophenyl) acetyl VH region, suggesting that the same VH germ-line might be used to encode two heavy chains contributing to antibodies of discrete specificities. A specific VH probe was derived and annealed to Eco RI and Bg1 II restriction fragments of liver (unrearranged) DNA extracted from the BALB/c, DBA/2 and C57BL/6 mouse strains that differ in their H chain allotypes. Under stringent conditions, only a few bands were identified by Southern blotting. The different patterns observed suggest that the VH anti-GAT repertoire differs between these strains even though their various anti-GAT antibodies express the same public idiotypic specificities.     

Predominance of nonproductive rearrangements of VH81X gene segments evidences a dependence of B cell clonal maturation on the structure of nascent H chains

Ig H chain V regions using the VH81X gene segment were PCR amplified from genomic DNA obtained from either splenic B cells or surface (s)Ig- bone marrow cells of BALB/c mice. Sequence analysis demonstrated that 93% of VH81X containing H chain V region genes in splenic B cells were rearranged nonproductively. Furthermore, 74% of rearrangements of VH81X among sIg- bone marrow cells were nonproductive. This contrasts with previous results obtained for rearrangements of members of the VH36-60 gene segment family among sIg- cells wherein, as a consequence of extensive clonal expansion after productive H chain V gene rearrangement, 80% of rearrangements were productive. The low proportion of productive rearrangements of VH81X is interpreted as indicating that most productive rearrangements of VH81X cannot facilitate clonal expansion, which would support the hypothesis that selection for clonal expansion and maturation is dependent on the amino acid sequence of nascent H chains. Additionally, because most productive rearrangements of VH81X cannot facilitate clonal maturation but do appear to mediate allelic exclusion, these processes are likely to be regulated independently.